Michael Anglesio

A Proteogenomic View of Synchronous Endometrioid Endometrial and Ovarian Cancer

Abstract Purpose: Increasing genomics-based evidence suggests that synchronous endometrial and ovarian cancer (SEOC) represents clonally related primary and metastatic tumors. A systematic analysis of the global protein landscape of SEOCs, heretofore lacking, could reveal functional and disease-specific consequences of known genetic alterations, the directionality of metastasis, and accurate histologic markers to distinguish SEOCs from single-site tumors. Experimental Design: We performed a systematic proteogenomic analysis of 29 patients diagnosed with SEOC at three international gynecologic oncology treatment centers (Chicago, Vancouver, and Tübingen). For direct comparison with single-site tumors, we included 9 patients with single-site endometrioid ovarian and 26 patients with single-site endometrioid endometrial cancer (EEC). For all 64 patients, we performed sequencing of a 275-gene cancer panel combined with compartment-resolved mass spectrometry–based proteomics of consecutive tissue sections to compare global (6,000+ proteins), tumor, and stromal proteomes. Results: DNA-based panel sequencing confirmed that most SEOCs are clonally related. Global proteome profiling uncovered pronounced differences between SEOCs and single tumors and underscored the importance of the stromal proteome in defining and identifying SEOCs. We identified molecularly unique SEOC stromal proteomes, which were globally more related to single endometrial cancers. We finally derived a proteomic predictor distinguishing SEOCs from single-site ovarian and uterine tumors. Conclusions: The integrated proteogenomic data show that SEOCs are distinguishable from endometrioid endometrial or endometrioid ovarian cancer. Based on their proteogenomic similarity to EECs, we conclude that most SEOCs represent primary EECs that have metastasized to the ovary.

Patterns of Associations with Epidemiologic Factors by High-Grade Serous Ovarian Cancer Gene Expression Subtypes

Abstract Background: Ovarian high-grade serous carcinomas (HGSC) comprise four distinct molecular subtypes based on mRNA expression patterns, with differential survival. Understanding risk factor associations is important to elucidate the etiology of HGSC. We investigated associations between different epidemiologic risk factors and HGSC molecular subtypes. Methods: We pooled data from 11 case–control studies with epidemiologic and tumor gene expression data from custom NanoString CodeSets developed through a collaboration within the Ovarian Tumor Tissue Analysis consortium. The PrOTYPE-validated NanoString-based 55-gene classifier was used to assign HGSC gene expression subtypes. We examined associations between epidemiologic factors and HGSC subtypes in 2,070 cases and 16,633 controls using multivariable-adjusted polytomous regression models. Results: Among the 2,070 HGSC cases, 556 (27%) were classified as C1.MES, 340 (16%) as C5.PRO, 538 (26%) as C2.IMM, and 636 (31%) as C4.DIF. The key factors, including oral contraceptive use, parity, breastfeeding, and family history of ovarian cancer, were similarly associated with all subtypes. Heterogeneity was observed for several factors. Former smoking [OR = 1.25; 95% confidence interval (CI) = 1.03, 1.51] and genital powder use (OR = 1.42; 95% CI = 1.08, 1.86) were uniquely associated with C2.IMM. History of endometriosis was associated with C5.PRO (OR = 1.46; 95% CI = 0.98, 2.16) and C4.DIF (OR = 1.27; 95% CI = 0.94, 1.71) only. Family history of breast cancer (OR = 1.44; 95% CI = 1.16, 1.78) and current smoking (OR = 1.40; 95% CI = 1.11, 1.76) were associated with C4.DIF only. Conclusions: This study observed heterogeneous associations of epidemiologic and modifiable factors with HGSC molecular subtypes. Impact: The different patterns of associations may provide key information about the etiology of the four subtypes.

Cellular origins of mucinous ovarian carcinoma

AbstractMucinous ovarian carcinoma (MOC) is a rare histotype of epithelial ovarian cancer. Its origins are obscure: while many mucinous tumours in the ovary are metastases from the gastrointestinal tract, MOC can occur as an ovarian primary; however, the cell of origin is not well established. In this review we summarise the pathological, epidemiological, and molecular evidence for the cellular origins of MOC. We propose a model for the origins of the various tumours of the ovary with mucinous differentiation. We distinguish Müllerian from gastrointestinal‐type mucinous differentiation. A small proportion of the latter arise from teratoma and a distinct terminology has been proposed. Other gastrointestinal mucinous tumours are associated with Brenner tumours and arise from their associated benign lesions, Walthard nests. The remaining mucinous tumours develop either through mucinous metaplasia in established Müllerian tumours or with even greater plasticity through gastrointestinal metaplasia of epithelial or mesothelial ovarian inclusions. This model remains to be validated and mechanistically understood and we discuss future research directions. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Cell State of Origin Impacts Development of Distinct Endometriosis-Related Ovarian Carcinoma Histotypes

Abstract Clear cell ovarian carcinoma (CCOC) and endometrioid ovarian carcinoma (ENOC) are ovarian carcinoma histotypes, which are both thought to arise from ectopic endometrial (or endometrial-like) cells through an endometriosis intermediate. How the same cell type of origin gives rise to two morphologically and biologically different histotypes has been perplexing, particularly given that recurrent genetic mutations are common to both and present in nonmalignant precursors. We used RNA transcription analysis to show that the expression profiles of CCOC and ENOC resemble those of normal endometrium at secretory and proliferative phases of the menstrual cycle, respectively. DNA methylation at the promoter of the estrogen receptor (ER) gene (ESR1) was enriched in CCOC, which could potentially lock the cells in the secretory state. Compared with normal secretory-type endometrium, CCOC was further defined by increased expression of cysteine and glutathione synthesis pathway genes and downregulation of the iron antiporter, suggesting iron addiction and highlighting ferroptosis as a potential therapeutic target. Overall, these findings suggest that while CCOC and ENOC arise from the same cell type, these histotypes likely originate from different cell states. This “cell state of origin” model may help to explain the presence of histologic and molecular cancer subtypes arising in other organs. Significance: Two cancer histotypes diverge from a common cell of origin epigenetically locked in different cell states, highlighting the importance of considering cell state to better understand the cell of origin of cancer.

p53 and ovarian carcinoma survival: an Ovarian Tumor Tissue Analysis consortium study

AbstractOur objective was to test whether p53 expression status is associated with survival for women diagnosed with the most common ovarian carcinoma histotypes (high‐grade serous carcinoma [HGSC], endometrioid carcinoma [EC], and clear cell carcinoma [CCC]) using a large multi‐institutional cohort from the Ovarian Tumor Tissue Analysis (OTTA) consortium. p53 expression was assessed on 6,678 cases represented on tissue microarrays from 25 participating OTTA study sites using a previously validated immunohistochemical (IHC) assay as a surrogate for the presence and functional effect of TP53 mutations. Three abnormal expression patterns (overexpression, complete absence, and cytoplasmic) and the normal (wild type) pattern were recorded. Survival analyses were performed by histotype. The frequency of abnormal p53 expression was 93.4% (4,630/4,957) in HGSC compared to 11.9% (116/973) in EC and 11.5% (86/748) in CCC. In HGSC, there were no differences in overall survival across the abnormal p53 expression patterns. However, in EC and CCC, abnormal p53 expression was associated with an increased risk of death for women diagnosed with EC in multivariate analysis compared to normal p53 as the reference (hazard ratio [HR] = 2.18, 95% confidence interval [CI] 1.36–3.47, p = 0.0011) and with CCC (HR = 1.57, 95% CI 1.11–2.22, p = 0.012). Abnormal p53 was also associated with shorter overall survival in The International Federation of Gynecology and Obstetrics stage I/II EC and CCC. Our study provides further evidence that functional groups of TP53 mutations assessed by abnormal surrogate p53 IHC patterns are not associated with survival in HGSC. In contrast, we validate that abnormal p53 IHC is a strong independent prognostic marker for EC and demonstrate for the first time an independent prognostic association of abnormal p53 IHC with overall survival in patients with CCC.

Selection of endometrial carcinomas for p53 immunohistochemistry based on nuclear features

AbstractThe World Health Organization endorses molecular subclassification of endometrial endometrioid carcinomas (EECs). Our objectives were to test the sensitivity of tumor morphology in capturing p53 abnormal (p53abn) cases and to model the impact of p53abn on changes to ESGO/ESTRO/ESP (European Society of Gynaecological Oncology/European Society for Radiotherapy and Oncology/European Society of Pathology) risk stratification. A total of 292 consecutive endometrial carcinoma resections received at Foothills Medical Centre, Calgary, Canada (2019–2021) were retrieved and assigned to ESGO risk groups with and without p53 status. Three pathologists reviewed the representative H&E‐stained slides, predicted the p53 status, and indicated whether p53 immunohistochemistry (IHC) would be ordered. Population‐based survival for endometrial carcinomas diagnosed during 2008–2016 in Alberta was obtained from the Alberta Cancer Registry. The cohort consisted mostly of grade 1/2 endometrioid carcinomas (EEC1/2; N = 218, 74.6%). One hundred and fifty‐two EEC1/2 (52.1% overall) were stage IA and 147 (50.3%) were low risk by ESGO. The overall prevalence of p53abn and subclonal p53 was 14.5 and 8.3%, respectively. The average sensitivity of predicting p53abn among observers was 83.6%. Observers requested p53 IHC for 39.4% with 98.5% sensitivity to detect p53abn (99.6% negative predictive value). Nuclear features including smudged chromatin, pleomorphism, atypical mitoses, and tumor giant cells accurately predicted p53abn. In 7/292 (2.4%), p53abn upgraded ESGO risk groups (2 to intermediate risk, 5 to high risk). EEC1/2/stage IA patients had an excellent disease‐specific 5‐year survival of 98.5%. Pathologists can select cases for p53 testing with high sensitivity and low risk of false negativity. Molecular characterization of endometrial carcinomas has great potential to refine ESGO risk classification for a small subset but offers little value for approximately half of endometrial carcinomas, namely, EEC1/2/stage IA cases.

Methylation Signature Implicated in Immuno-Suppressive Activities in Tubo-Ovarian High-Grade Serous Carcinoma

Abstract Background: Better understanding of prognostic factors in tubo-ovarian high-grade serous carcinoma (HGSC) is critical, as diagnosis confers an aggressive disease course. Variation in tumor DNA methylation shows promise predicting outcome, yet prior studies were largely platform-specific and unable to evaluate multiple molecular features. Methods: We analyzed genome-wide DNA methylation in 1,040 frozen HGSC, including 325 previously reported upon, seeking a multi-platform quantitative methylation signature that we evaluated in relation to clinical features, tumor characteristics, time to recurrence/death, extent of CD8+ tumor-infiltrating lymphocytes (TIL), gene expression molecular subtypes, and gene expression of the ATP-binding cassette transporter TAP1. Results: Methylation signature was associated with shorter time to recurrence, independent of clinical factors (N = 715 new set, hazard ratio (HR), 1.65; 95% confidence interval (CI), 1.10–2.46; P = 0.015; N = 325 published set HR, 2.87; 95% CI, 2.17–3.81; P = 2.2 × 10−13) and remained prognostic after adjustment for gene expression molecular subtype and TAP1 expression (N = 599; HR, 2.22; 95% CI, 1.66–2.95; P = 4.1 × 10−8). Methylation signature was inversely related to CD8+ TIL levels (P = 2.4 × 10−7) and TAP1 expression (P = 0.0011) and was associated with gene expression molecular subtype (P = 5.9 × 10−4) in covariate-adjusted analysis. Conclusions: Multi-center analysis identified a novel quantitative tumor methylation signature of HGSC applicable to numerous commercially available platforms indicative of shorter time to recurrence/death, adjusting for other factors. Along with immune cell composition analysis, these results suggest a role for DNA methylation in the immunosuppressive microenvironment. Impact: This work aids in identification of targetable epigenome processes and stratification of patients for whom tailored treatment may be most beneficial.

Development and Validation of the Gene Expression Predictor of High-grade Serous Ovarian Carcinoma Molecular SubTYPE (PrOTYPE)

Abstract Purpose: Gene expression–based molecular subtypes of high-grade serous tubo-ovarian cancer (HGSOC), demonstrated across multiple studies, may provide improved stratification for molecularly targeted trials. However, evaluation of clinical utility has been hindered by nonstandardized methods, which are not applicable in a clinical setting. We sought to generate a clinical grade minimal gene set assay for classification of individual tumor specimens into HGSOC subtypes and confirm previously reported subtype-associated features. Experimental Design: Adopting two independent approaches, we derived and internally validated algorithms for subtype prediction using published gene expression data from 1,650 tumors. We applied resulting models to NanoString data on 3,829 HGSOCs from the Ovarian Tumor Tissue Analysis consortium. We further developed, confirmed, and validated a reduced, minimal gene set predictor, with methods suitable for a single-patient setting. Results: Gene expression data were used to derive the predictor of high-grade serous ovarian carcinoma molecular subtype (PrOTYPE) assay. We established a de facto standard as a consensus of two parallel approaches. PrOTYPE subtypes are significantly associated with age, stage, residual disease, tumor-infiltrating lymphocytes, and outcome. The locked-down clinical grade PrOTYPE test includes a model with 55 genes that predicted gene expression subtype with >95% accuracy that was maintained in all analytic and biological validations. Conclusions: We validated the PrOTYPE assay following the Institute of Medicine guidelines for the development of omics-based tests. This fully defined and locked-down clinical grade assay will enable trial design with molecular subtype stratification and allow for objective assessment of the predictive value of HGSOC molecular subtypes in precision medicine applications. See related commentary by McMullen et al., p. 5271

The proteome of clear cell ovarian carcinoma

AbstractClear cell ovarian carcinoma (CCOC) is the second most common subtype of epithelial ovarian carcinoma. Late‐stage CCOC is not responsive to gold‐standard chemotherapy and results in suboptimal outcomes for patients. In‐depth molecular insight is urgently needed to stratify the disease and drive therapeutic development. We conducted global proteomics for 192 cases of CCOC and compared these with other epithelial ovarian carcinoma subtypes. Our results showed distinct proteomic differences in CCOC compared with other epithelial ovarian cancer subtypes including alterations in lipid and purine metabolism pathways. Furthermore, we report potential clinically significant proteomic subgroups within CCOC, suggesting the biologic plausibility of stratified treatment for this cancer. Taken together, our results provide a comprehensive understanding of the CCOC proteomic landscape to facilitate future understanding and research of this disease. © 2022 The Pathological Society of Great Britain and Ireland.

Molecular Subclasses of Clear Cell Ovarian Carcinoma and Their Impact on Disease Behavior and Outcomes

Abstract Purpose: To identify molecular subclasses of clear cell ovarian carcinoma (CCOC) and assess their impact on clinical presentation and outcomes. Experimental Design: We profiled 421 primary CCOCs that passed quality control using a targeted deep sequencing panel of 163 putative CCOC driver genes and whole transcriptome sequencing of 211 of these tumors. Molecularly defined subgroups were identified and tested for association with clinical characteristics and overall survival. Results: We detected a putative somatic driver mutation in at least one candidate gene in 95% (401/421) of CCOC tumors including ARID1A (in 49% of tumors), PIK3CA (49%), TERT (20%), and TP53 (16%). Clustering of cancer driver mutations and RNA expression converged upon two distinct subclasses of CCOC. The first was dominated by ARID1A-mutated tumors with enriched expression of canonical CCOC genes and markers of platinum resistance; the second was largely comprised of tumors with TP53 mutations and enriched for the expression of genes involved in extracellular matrix organization and mesenchymal differentiation. Compared with the ARID1A-mutated group, women with TP53-mutated tumors were more likely to have advanced-stage disease, no antecedent history of endometriosis, and poorer survival, driven by their advanced stage at presentation. In women with ARID1A-mutated tumors, there was a trend toward a lower rate of response to first-line platinum-based therapy. Conclusions: Our study suggests that CCOC consists of two distinct molecular subclasses with distinct clinical presentation and outcomes, with potential relevance to both traditional and experimental therapy responsiveness. See related commentary by Lheureux, p. 4838

Validated biomarker assays confirm that ARID1A loss is confounded with MMR deficiency, CD8 + TIL infiltration, and provides no independent prognostic value in endometriosis‐associated ovarian carcinomas

Abstract ARID1A (BAF250a) is a component of the SWI/SNF chromatin modifying complex, plays an important tumour suppressor role, and is considered prognostic in several malignancies. However, in ovarian carcinomas there are contradictory reports on its relationship to outcome, immune response, and correlation with clinicopathological features. We assembled a series of 1623 endometriosis‐associated ovarian carcinomas, including 1078 endometrioid (ENOC) and 545 clear cell (CCOC) ovarian carcinomas, through combining resources of the Ovarian Tumor Tissue Analysis (OTTA) Consortium, the Canadian Ovarian Unified Experimental Resource (COEUR), local, and collaborative networks. Validated immunohistochemical surrogate assays for ARID1A mutations were applied to all samples. We investigated associations between ARID1A loss/mutation, clinical features, outcome, CD8 + tumour‐infiltrating lymphocytes (CD8 + TILs), and DNA mismatch repair deficiency (MMRd). ARID1A loss was observed in 42% of CCOCs and 25% of ENOCs. We found no associations between ARID1A loss and outcomes, stage, age, or CD8 + TIL status in CCOC. Similarly, we found no association with outcome or stage in endometrioid cases. In ENOC, ARID1A loss was more prevalent in younger patients ( p  = 0.012) and was associated with MMRd ( p  < 0.001) and the presence of CD8 + TILs ( p  = 0.008). Consistent with MMRd being causative of ARID1A mutations, in a subset of ENOCs we also observed an association with ARID1A loss‐of‐function mutation as a result of small indels ( p  = 0.035, versus single nucleotide variants). In ENOC, the association with ARID1A loss, CD8 + TILs, and age appears confounded by MMRd status. Although this observation does not explicitly rule out a role for ARID1A influence on CD8 + TIL infiltration in ENOC, given current knowledge regarding MMRd, it seems more likely that effects are dominated by the hypermutation phenotype. This large dataset with consistently applied biomarker assessment now provides a benchmark for the prevalence of ARID1A loss‐of‐function mutations in endometriosis‐associated ovarian cancers and brings clarity to the prognostic significance. © 2021 The Pathological Society of Great Britain and Ireland.

Concurrent RB1 Loss and BRCA Deficiency Predicts Enhanced Immunologic Response and Long-term Survival in Tubo-ovarian High-grade Serous Carcinoma

Abstract Purpose: The purpose of this study was to evaluate RB1 expression and survival across ovarian carcinoma histotypes and how co-occurrence of BRCA1 or BRCA2 (BRCA) alterations and RB1 loss influences survival in tubo-ovarian high-grade serous carcinoma (HGSC). Experimental Design: RB1 protein expression was classified by immunohistochemistry in ovarian carcinomas of 7,436 patients from the Ovarian Tumor Tissue Analysis consortium. We examined RB1 expression and germline BRCA status in a subset of 1,134 HGSC, and related genotype to overall survival (OS), tumor-infiltrating CD8+ lymphocytes, and transcriptomic subtypes. Using CRISPR-Cas9, we deleted RB1 in HGSC cells with and without BRCA1 alterations to model co-loss with treatment response. We performed whole-genome and transcriptome data analyses on 126 patients with primary HGSC to characterize tumors with concurrent BRCA deficiency and RB1 loss. Results: RB1 loss was associated with longer OS in HGSC but with poorer prognosis in endometrioid ovarian carcinoma. Patients with HGSC harboring both RB1 loss and pathogenic germline BRCA variants had superior OS compared with patients with either alteration alone, and their median OS was three times longer than those without pathogenic BRCA variants and retained RB1 expression (9.3 vs. 3.1 years). Enhanced sensitivity to cisplatin and paclitaxel was seen in BRCA1-altered cells with RB1 knockout. Combined RB1 loss and BRCA deficiency correlated with transcriptional markers of enhanced IFN response, cell-cycle deregulation, and reduced epithelial–mesenchymal transition. CD8+ lymphocytes were most prevalent in BRCA-deficient HGSC with co-loss of RB1. Conclusions: Co-occurrence of RB1 loss and BRCA deficiency was associated with exceptionally long survival in patients with HGSC, potentially due to better treatment response and immune stimulation.